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Effect of different cloning strategies in pET-28a on solubility and functionality of a staphylococcal phage endolysin

Hong Y Tham1, Adelene A-L Song1,2, Khatijah Yusoff1,2

  • 1Department of Microbiology, Faculty of Biotechnology & Biomolecular Sciences, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia.

Biotechniques
|August 14, 2020
PubMed

Insights

Cloning strategies impact endolysin functionality. Careful selection of cloning methods is crucial for producing functional endolysins, which are potential antibiotic alternatives against methicillin-resistant Staphylococcus aureus (MRSA).

Area of Science:

  • Microbiology
  • Biotechnology
  • Protein Engineering

Background:

  • Endolysins are phage-derived proteins investigated as alternatives to conventional antibiotics.
  • Methicillin-resistant Staphylococcus aureus (MRSA) poses a significant public health threat, necessitating novel therapeutic approaches.

Purpose of the Study:

  • To clone and express a functional endolysin targeting MRSA.
  • To evaluate the impact of different cloning strategies on endolysin activity.

Main Methods:

  • Endolysin gene from an MRSA-infecting phage was cloned into Escherichia coli pET28a expression vector.
  • Two distinct cloning strategies using different restriction enzymes (BamHI/XhoI vs. NcoI/XhoI) were employed.
  • Protein expression, solubility, and functionality (halo zone formation, turbidity reduction) were assessed.

Main Results:

  • The initial cloning strategy (BamHI/XhoI) yielded insoluble, nonfunctional endolysin, despite successful solubilization.
  • A modified cloning strategy (NcoI/XhoI) at 18°C resulted in soluble and functional endolysin.
  • The functional endolysin demonstrated lytic activity against MRSA, indicated by halo zone formation and reduced bacterial turbidity.

Conclusions:

  • Cloning strategy significantly influences the functional expression of endolysins.
  • Careful optimization of cloning techniques is essential for developing effective endolysin-based therapeutics.
  • This study highlights the importance of considering cloning methods for successful protein expression and application.
Keywords:
MRSAantimicrobial activityendolysininclusion bodypET28aprotein expression

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